This project will determine the effect on mutation frequency of repair of DNA allowing alkylation of DNA in oocytes of Drosophila melanogaster. Environmental mutagens exhibit considerable variance in the frequency of mutations induced in different germ cell stages. The role which repair of DNA plays in determining this variation, particularly in oocytes, will be studied by comparing in repair competent and repair deficient strains of D. melanogaster four responses to treatment with model mutagens: 1) the initial alkylation level; 2) the change in alkylation levels and patterns over time; 3) levels of unscheduled DNA synthesis (UDS); and 4) induced mutation frequency in the sex-linked recessive lethal test. Previous published work on in vivo molecular dosimetry in germ cells has been limited to postmeiotic germ cell stages in the male. Oocytes provide both a new model system for basic research and, because they are metabolically active and represent the germ cell stage most exposed in humans, a more relevant system for risk estimation than post meiotic male germ cells. Research on this project has developed methods for in vivo molecular dosimetry based on adduct formation and UDS in the developing oocyte of female D. melanogaster. This has involved developing methods for separating the high molecular weight of nuclear DNA from mitochondrial DNA, RNA, and low molecular weight fragments of nucleic acid. Our research has shown that the high molecular weight nuclear DNA is conserved from the last nuclear DNA scheduled synthesis in the germarium until cleavage in the egg, thus providing the complete range of oocyte development for UDS and molecular dosimetry determination. It has also been shown that oocytes treated in the female and analyzed after the egg is laid exhibit DNA repair. D. melanogaster strains that are homozygous for the excision deficient mutant mei-9L exhibit less UDS and retain adducts longer than repair competent strains; furthermore, the homozygous mei-9L strain has a higher mutation frequency following treatment of oocytes with EMS. The experimental system will be manipulated using a variety of alkylating agents and repair deficient strains. Genetic tests performed parallel to the in vivo molecular dosimetry are conducted using the sex-linked recessive lethal test that has been modified for use in treated females.